Lepidopteran insect cell substrates for recombinant protein production include Spodoptera frugiperda IPLB-SF21-AE (Sf21), its more commonly used Sf9 clonal isolate, and various Trichoplusia ni cell lines, such as High Five. These cell lines are utilized in the biotechnology industry as host cells in the Baculovirus Expression Vector System (BEVS) for recombinant protein production, or are transfected or transformed with a plasmid DNA expression vector to transiently or stably express a protein of interest (Richardson, C. D. (Ed.), Baculovirus Expression Protocols. Methods in Molecular Biology 39:65-202 (1995)).
Sf21 and Sf9 are commonly cultured in commercially available serum-free media formulations which utilize a phosphate buffer system to maintain a culture pH in the optimal range of 6.0-6.4 (Licari et al. Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity. Biotechnology Progress 9: 146-152 (1993) and Drugmand et al. Insect cells as factories for biomanufacturing. Biotechnology Advances 30:1140-1157 (2012)) for both cultivation and recombinant protein production. A broader pH requirement of 6.0-6.8 for various insect cell lines has also been previously described, with deleterious effects on cell growth and viability reported upon small deviations outside of this range (Drugmand et al., supra). While the normal culture pH range is suitable for the production of a variety of recombinant proteins, some target proteins cannot be produced effectively in their desired form under these conditions. Viral glycoproteins involved in fusion, often used as vaccine antigens, are one example due to pH-sensitive conformational changes which occur at a threshold pH in the range of typical insect cell culture.
Virions from the alphavirus genus contain structural E1 and E2 glycoproteins with a pH threshold for structure conformational change in the range of typical insect cell culture (Lee et al. (2011), supra; Li et al. (2010), supra). Despite this, Sindbis virus has been shown to replicate and produce functional, infectious virions in Sf21 cells when cultured under standard conditions (Hafer et al., Differential incorporation of cholesterol by Sindbis virus grown in mammalian or insect cells. J. Virol. 83(18):9113-9121 (2009); and Wang et al. Infection of cells by Sindbis virus at low temperature. Virology 362: 461-467 (2007)). Pijlman et al. (WO 2012/130723) report the expression of salmonid alphavirus VLPs in insect cells. Chikungunya virus (CHIKV) is a closely related virus for which recombinant virus like particles (VLPs) have been produced in mammalian cell culture (Akahata et al., A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection. Nature Medicine 16(3):334-338 (2010)), but when a similar transgene was delivered via baculovirus infection in Sf21, expression of multiple CHIKV proteins was reported without mention of VLP production (Kuo et al. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion. J. Biomedical Science 19:44 (2012)).